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Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

DOI: 10.1107/S2052252521001299 DOI Help

Authors: Joao Ramos (Institut Laue-Langevin; Partnership for Structural Biology (PSB); University of Copenhagen) , Valerie Laux (Institut Laue–Langevin; Partnership for Structural Biology (PSB)) , Michael Haertlein (Institut Laue–Langevin; Partnership for Structural Biology (PSB)) , Elisabetta Boeri Erba (Institut Laue–Langevin; Partnership for Structural Biology (PSB)) , Katherine E. Mcauley (Diamond Light Source; Paul Scherrer Institute) , V. Trevor Forsyth (Institut Laue–Langevin; Partnership for Structural Biology (PSB); Keele University) , Estelle Mossou (Institut Laue–Langevin; Partnership for Structural Biology (PSB); Keele University) , Sine Larsen (University of Copenhagen) , Annette E. Langkilde (University of Copenhagen)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 8

State: Published (Approved)
Published: May 2021
Diamond Proposal Number(s): 24717

Open Access Open Access

Abstract: This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

Journal Keywords: perdeuterated lysozyme; HEWL; isotope effect; protein refolding; biophysical characterization; X-ray crystallography; protein structure; structural biology.

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

Other Facilities: BioMAX at MAX IV

Documents:
jt5055.pdf

Discipline Tags:

Life Sciences & Biotech Structural biology

Technical Tags:

Diffraction Macromolecular Crystallography (MX)